A novel fluorescent probe with a phosphofluorene molecular structure for selective detection of hydrogen sulfide in living cells

Hydrogen sulfide (H2S) gas plays a significant role in biological regulation. With advancements in technology, H2S has been discovered across diverse fields, necessitating a comprehensive understanding of its physiological functions through monitoring changes in H2S within complex environments and physiological processes. In this study, we designed a phosphofluorene-based conjugate probe PPF-CDNB with an asymmetric π-conjugated phosphine structure and utilized dinitrophenyl ether as the recognition site for H2S. PPF-CDNB exhibited exceptional resistance to interference and demonstrated stability over a broad pH range (3.0–10.0), making it suitable for various environmental conditions. Intracellular experiments revealed that PPF-CDNB effectively monitored both endogenous and exogenous levels of H2S.


Introduction
Hydrogen sulde (H 2 S) is a gaseous compound characterized by an unpleasant odor reminiscent of rotten eggs.][10] Elevated levels of H 2 S have been associated with various conditions including Alzheimer's disease, Down syndrome, Parkinson's disease, and diabetes. 11,12Hydrogen sulde represents a bioactive molecule that assumes critical regulatory functions within biological systems. 13,14However, aberrant levels of H 2 S are closely associated with functional impairments and various diseases, prompting active development of small molecule chemical tools for investigating its diverse roles in biology and medicine. 15In addition to its lethal effects, H 2 S also modulates a wide range of physiological actions including vasodilation, anti-inammatory effects, insulin release, neurotransmission, antioxidant properties, anti-apoptotic effects, and neuroprotection. 16Notably, H 2 S triggers S-sulydration of Keap1, activating Nrf 2 and facilitating its nuclear translocation, which results in the production of antioxidant proteins. 17Numerous studies have demonstrated that H 2 S ranks as the third most crucial signaling molecule in organisms following CO and NO. 18,191][22] Thus, there is a pressing need for simple and rapid detection methods to measure H 2 S concentration accurately in various environments, including water phase, gas phase, and living cells (both exogenous and endogenous).Among the available detection methods, the uorescence probe detection method has gained signicant attention due to its high sensitivity, selectivity, cost-effectiveness, ease of operation, and real-time determination.
8][29][30][31] Similar to uorene, PPF exhibits a wide energy gap, and its uorescence spectrum is predominantly observed in the ultraviolet region (366 nm). 32,33The presence of two lateral benzene rings in PPF provides the system with several advantages over monocyclic phosphoheterocycles in binding molecular electronics. 34,35The synthesis and operation of the tricyclic system can modulate the overall electronic structure through the introduction and manipulation of benzene ring substituents. 36,379][40][41] Despite these advantages, the use of PPF structures in uorescent probes is relatively uncommon.Most commercially available H 2 S uorescent probes rely on reduction and nucleophilic reactions for detection, which suffer from slow reaction rates leading to inef-cient detection processes lasting tens of minutes or even hours.For instance, Kim's group designed the uorescent probe BT-ITC, which showed emission at 447 nm and had a response time of 40 min to H 2 S. 42 Fang's group designed the uorescent probe Mito-GW, which has a response time of 80 min to H 2 S and displays emission at 447 nm. 43Zhong's group designed a uorescent probe, THQ-L, that displayed emission at 650 nm with a response time of 45 min to H 2 S. 44 While these results contribute to the advancement of uorescent probes, they also have limitations, notably long response times (exceeding 20 minutes).Therefore, there is a critical need to design a uorescent probe with rapid response, high selectivity, and sensitivity to H 2 S detection.In response to this need, we designed and synthesized a uorescent probe, PPF-CDNB.The phosphom-uorene derivative of the probe was used as a uorophore, while 2,4-dinitrophenyl was employed as a functional recognition group.PPF-CDNB exhibits a wide pH range (3.0-10.0),excellent sensitivity (LOD = 150 nM), and high selectivity for H 2 S detection, minimizing potential interference.Thus, PPF-CDNB offers a fast and efficient method for detecting H 2 S.

Instruments and reagents
All reagents and solvents were purchased from commercial suppliers and used without further purication.Distilled water was utilized in the experiment aer passing through a water superpurication system.Silica column chromatography was conducted using 200-300 mesh silica and an appropriate solvent.The reaction progress was monitored by thin-layer chromatography (TLC), and the reaction components were visualized using UV light.Fluorescence spectra and relative uorescence intensities were measured using the Shimadzu RF-5301 uorescence spectrometer.The excitation wavelength for all uorescence measurements was 294 nm, with excitation and emission slit widths set to 2.5 nm.Ultraviolet-visible spectroscopy was performed using the Shimadzu UV-2700 spectrophotometer.The 1 H-NMR and 13 C-NMR spectra were recorded on the BRUKER600 spectrometer.pH measurements were conducted using a PHS-3C pH meter.Electrospray mass spectrometry (ESI-MS) data were acquired using the Agilent 1100 series instrument.Cell images were captured using a uorescence microscope (Leica, Germany).

Synthesis of probe PPF-CDNB
PPF-OH (200 mg, 0.68 mmol) and 2,4-dinitrophenyl chloride (166.4 mg, 0.82 mmol) were dissolved in 7 mL DMF and added to a round-bottom ask.Potassium carbonate (141 mg, 1.03 mmol) was then added, and stirred at 100 °C for 6 h.Aer the reaction, the mixture was extracted with ethyl acetate, and the ethyl acetate extract was passed through a column.The column was eluted with a mixture of ethyl acetate and petroleum ether to yield a yellow solid compound, PPF-CDNB (267 mg, 85.6%) (Fig. 1).

Fluorometric measurements
To further investigate the performance of the probe, spectral experiments were conducted.Initially, a detection solution was prepared by weighing an appropriate amount of probe PPF-CDNB and dissolving it in DMF solution to create a 1 mM stock solution.Subsequently, 100 mL of the probe stock solution was added to a volumetric ask containing PBS/DMF (v/v = 9 : 1, pH = 7.4) buffer solution to achieve a concentration of 20 mM.Spectral measurements were then conducted in both the presence and absence of analytes Typically, the reaction between probe PPF-CDNB and NaHS occurs within the PBS/DMF (v/v = 9 : 1, pH = 7.4) system followed by measuring the uorescence intensity at 528 nm in the reaction solution.

Selectivity and specicity
The probe stock solution (100 mL) was prepared in DMF.Various test substances (K + , Cu + , Ca 2+ , Na + , I Cys, D-Cys, GSH, Zn 2+ ) were prepared in distilled water.All anions are prepared from their sodium salts, and all cations are prepared from their chloride salts.The resulting solution was stored at room temperature (25 °C), and then the uorescence spectrum was recorded.

Cell cultures and imaging
For exogenous imaging, A549 cells were incubated with PPF-CDNB (10 mM) at 37 °C for 30 minutes and then cultured with NaHS (50 mM) for 30 minutes.For endogenous imaging, the A549 cells were divided into three plates.One plate was incubated with cysteine (Cys, 50 mM) and PPF-CDNB (10 mM) for 30 minutes.The second plate was incubated with DL-propyl glycine (PAG, 100 mM) and PPF-CDNB (10 mM) for 30 min, and the third plate was incubated with DL-propyl glycine (PPG, 100 mM), cysteine (Cys, 50 mM) and PPF-CDNB (10 mM) for 30 min.The images were obtained under a uorescence microscope.

Photophysical properties of the PPF-CDNB probe
For probe PPF-CDNB, 10% DMF was added to PBS buffer solution to enhance its solubility and uorescence (Fig. S1 †).We investigated the interaction of probe PPF-CDNB (20 mM) with H 2 S in a PBS/DMF (V/V = 9/1, pH 7.4) system by UV absorption spectroscopy.As shown in the Fig. 2, the maximum UV absorption of PPF-CDNB probe is located at 294 nm.By increasing the concentration of PPF-CDNB probe (20-100 mM) (Fig. 2a), the absorbance coefficient at 294 nm increases linearly, according to the linear regression equation (R 2 = 0.975) (Fig. 2b).When the PPF-CDNB probe concentration was 20 mM, the absorbance at 294 nm was obviously decreased and the UV absorbance at 406 nm was signicantly increased by adding NaHS solution (0-10 eq.) (Fig. 2c).The color of the solution changed from colorless (Fig. 2d(I)) to brown-yellow (Fig. 2d(II)), and new compounds were formed.

Effect of pH on H 2 S recognition by the PPF-CDNB probe
For successful bioluminescence imaging, the PPF-CDNB probe must be activated within the appropriate physiological pH range to investigate the effects of different pH conditions on its performance (Fig. 3d).It was observed that the PPF-CDNB probe was basically unaffected by pH in the pH range of 4.0-10.0.

RSC Advances Paper
However, upon addition of H 2 S, the uorescence intensity of the PPF-CDNB probe at 528 nm signicantly increased.Within the pH range of 1.0-4.0,as the acidity increases, the uorescence intensity of both PPF-CDNB probe and PPF-CDNB probe with H 2 S signicantly decreases (Fig. S2 †).It is speculated that the structure of the PPF of the PPF-CDNB probe is disrupted, which disrupts the ESIPT process in the PPF uorophore.In contrast, within the pH range of 10.0-13.0, the uorescence intensity of both the PPF-CDNB probe and the PPF-CDNB probe with H 2 S increased signicantly with increasing alkalinity (Fig. S2 †).It is speculated that disruption in the structure of the 2,4-dinitrophenyl ether moiety blocked the PET process, leading to restoration of uorophore uorescence.These ndings indicate that the probe's response to H 2 S spans across acidic and alkaline conditions, enabling its applicability in various environments such as water phase, gas phase, and live cell measurements.This broad response range enhances its utility and value.

The selectivity and competitiveness of PPF-CDNB probe for H 2 S
The detection system oen comprises a variety of chemical substances, potentially interfering with the identication of target products.Therefore, the selectivity of PPF-CDNB probe with various ions was studied in PBS/DMF (V/V = 9/1, pH 7. the uorescence spectra at 528 nm were measured aer 10 minutes of incubation.Fluorescence spectra at 528 nm aer 10 min of action (Fig. 3a and b).The addition of organic salt and inorganic salt had no signicant effect on the uorescence intensity of PPF-CDNB probe.Although the addition of L-Cys and D-Cys can enhance the uorescence intensity of PPF-CDNB probe system, it was signicantly lower than that caused by the addition of NaHS (Fig. 3c).Notably, when interfering ions were added to the probe, the uorescence intensity was greatly enhanced upon the addition of NaHS, reaching a level comparable to the uorescence intensity without interfering ions.Therefore, the PPF-CDNB probe exhibits good selectivity for H 2 S detection. 47The detection limit (LOD) of the uorescence probe was calculated to be 0.15 mM (LOD = 3s/k) aer analysis (Fig. S3 †).The PPF-CDNB probe can accurately recognize H 2 S even under very harsh conditions.

Calculation of DFT
To comprehensively understand the uorescence change mechanism of probes PPF-CDNB and PPF-OH, DFT calculations were performed using the B3LYP/6-311g(d) level in the Gauss 09

Study on the kinetics of H 2 S by PPF-CDNB probe
UV-visible and uorescence titration experiments were conducted in PBS/DMF (V/V = 9/1, pH 7.4) buffer solution.As depicted in the gures, the uorescence intensity at 528 nm gradually increased with the addition of NaHS, becoming notably enhanced at 2 equivalents (Fig. 5a and b).Concurrently, the solution transitioned from colorless to yellow, emitting strong yellow uorescence under a 365 nm UV lamp (Fig. 5(I, II) S5 †).We also observed a time-dependent phenomenon with the PPF-CDNB probe (Fig. 5c and d).In the PBS/DMF system (V/V = 9/1, pH = 7.4), at a concentration of 20 mM, the uorescence intensity of PPF-CDNB gradually rose within the rst 10 minutes, reaching its peak at 10 minutes, and then stabilized.This experiment demonstrated that the uorescence intensity of PPF-CDNB exhibited a linear relationship with the concentration of NaHS in the range of 0-2 equivalents, con-rming the utility of PPF-CDNB as a tool for H 2 S detection (Fig. 5b).

Sensing mechanism
To further elucidate the reaction mechanism between PPF-CDNB and H 2 S, we conducted a 1 H-NMR titration experiment.Conrmation of the conversion of PPF-CDNB to PPF-OH was achieved through 1 H-NMR titration of PPF-CDNB in the presence of H 2 S in a DMSO-d6 solution (Fig. 6(II)).It was observed that the peak of 2,4-dinitrobenzene on PPF-CDNB gradually decreased with the increase of NaHS aer the addition of 1 eq., 3 eq., 5 eq., and 10 eq.NaHS, respectively.Following the addition of 5 eq.NaHS, the bimodal proton signal at 8.89 ppm, 8.47 ppm, 8.27 ppm, and 8.16 ppm disappeared.It moved to the  high-eld region again, appearing at 8.07 ppm, 7.60-7.62ppm and 7.33 ppm.Upon adding 10 equivalents of NaHS, the peak position remained essentially unchanged compared to adding 5 equivalents of NaHS, indicating that excess H 2 S did not affect the thiolysis reaction of PPF-CDNB.These ndings support a sensing mechanism involving hydrogen sulde-induced ether bond thiolysis (Fig. 6(I)).Additionally, the sensing mechanism was corroborated by HPLC analysis (Fig. S6 †).

Live cell uorescence imaging
Inspired by the above experimental results, we further investigated the practicality of probes for detecting intracellular H 2 S. Initially, we conducted standard studies to evaluate the cytotoxicity of the probes.In preliminary experiments, the cytotoxicity of PPF-CDNB on A549 cells was examined using a CCK-8 assay (Fig. S7 †).Our observations revealed that when treated with 10 mM PPF-CDNB for 12 hours, approximately 80% of A549 cells remained viable.The results indicate that the probe exhibited low toxicity to A549 cells (Fig. S7 †).
We utilized PPF-CDNB to detect both exogenous and endogenous H 2 S in living A549 cells, selecting a concentration of 10 mM based on cytotoxicity assays.Exogenous H 2 S detection was divided into two groups.In the rst group, A549 cells were pre-incubated with PPF-CDNB for 30 minutes as a blank control, resulting in minimal uorescence under uorescence microscopy (Fig. 7b).In the second group, A549 cells were preincubated with PPF-CDNB followed by 50 mM NaHS, leading to pronounced green uorescence in the cytoplasmic region of the cells (Fig. 7e).Demonstrate the decomposition of PPF-CDNB by exogenous H 2 S into PPF-OH, which emits dazzling uorescence.Light eld images of A549 cells incubated with PPF-CDNB and PPF-CDNB + H 2 S (Fig. 7a and d) showed no morphological changes in the cell structure, indicating the effectiveness of PPF-CDNB in detecting exogenous H 2 S.
The enzymes 3-mercaptopyruvate thiotransferase (3MST), cystathionine g-lyase (CSE), and cystathionine b-synthase (CBS) present in the cytoplasm utilize Cys as a substrate to produce endogenous H 2 S. 48 PPG is a potent H 2 S inhibitor of cystathionine g-lyase (CSE) synthesis.For endogenous H 2 S detection, experiments were divided into three groups.In the rst group, A549 cells were incubated with PPF-CDNB (10 mM) followed by the addition of cysteine (Cys) (50 mM), resulting in observable green uorescence (Fig. 7h).As a control in the second group, A549 cells pretreated with PPF-CDNB (10 mM) were incubated with only 200 mM PPG, no uorescence (Fig. 7k).In the third group, A549 cells were rst incubated with PPF-CDNB (10 mM), followed by the addition of Cys (50 mM) and 200 mM PPG before imaging under light microscopy, conrming the absence of green uorescence in the cytoplasm (Fig. 7n).
This highlights the role of PPG in inhibiting intracellular enzymes, thus preventing endogenous H 2 S production.In the absence of H 2 S in 2,4 dinitrobenzene ether will not cracking, PPF-CDNB remain inactive forms (no uorescence).Bright-eld images of A549 cells incubated with PPF-CDNB, PPF-CDNB + Cys, PPF-CDNB+ Cys + PPG, and PPF-CDNB + PPG showed no change in the morphology of the cellular structures (Fig. 7).It should be noted that the weak uorescence observed in PPF-CDNB treated A549 cells (Fig. 7b) may be due to the reaction of PPF-CDNB with a small amount of endogenous H 2 S present in the cytoplasm.Show that the PPF-CDNB endogenous H 2 S level is capable of detecting living cells.

Conclusions
We have established a novel molecular design strategy for uorescent probes, which can target H 2 S and exhibit signicant uorescence enhancement.This design is based on asymmetric p-conjugated phosphine oxide, achieved through the creation of the large p-bond conjugated probe PPF-CDNB with a PPF structure.The diaryl ether structure on the synthesized PPF compound showed a blue shi of more than 100 nm when thiolated (Fig. S4a †), which could signicantly reduce the interference of crosstalk signal and improve the accuracy of ratio measurement.Utilizing PPF-OH as a scaffold, we developed the PPF-CDNB uorescence probe, which markedly amplied the uorescence intensity of the H 2 S thiolysis reaction.Based on the complex environment in vivo, the selectivity experiment and anti-interference experiment of the probe were also conducted (Fig. 3b and c).These experiments revealed the probe's ability to distinguish H 2 S from other interfering substances, showcasing high selectivity and sensitivity.We believe that using this unique method of structural shi of PPF provides a exible platform for the design of various uorescent probes.
The rapid transformation of the probe in the green channel observed during live cell imaging indicates the cellular response of PPF-CDNB to H 2 S. Given its relevance to a myriad of biological phenomena and conditions, including Alzheimer's disease and down syndrome, PPF-CDNB is expected to serve as a valuable research tool for detecting related biological processes and diseases.

Fig. 3
Fig. 3 (a) Fluorescence spectra of probe PPF-CDNB (20.0 mM) after adding 22 analytes of 200 mM.(b) Bar chart showing the selectivity of other analytes to probe H 2 S detection.(c) Bar chart showing the anti-interference of other analytes to probe H 2 S detection.(d) Fluorescence intensity of 20 mM probe PPF-CDNB at different pH values the pH was adjusted with NaOH and HCl, and the fluorescence intensity at 528 nm at different pH values.In a buffer solution system of PBS/DMF (V/V = 9/1, pH 7.4).

Fig. 5
Fig. 5 Fluorescence titration curve of probe PPF-CDNB was obtained.(a) The fluorescence emission spectrum of 20 mM probe PPF-CDNB was recorded at 15 minutes after the addition of NaHS concentration (0-10 eq.).(b) A linear relationship between the fluorescence intensity of probe PPF-CDNB (20 mM) at 528 nm and NaHS concentration (0-2 eq.) was observed.(c) The fluorescence emission spectra of the 20 mM probe PPF-CDNB were measured at 10 eq.NaHS for 0-15 minutes.(d) Curve of fluorescence intensity of probe PPF-CDNB (20 mM) versus the time gradient between 0 and 15 min in the presence of 10 eq.NaHS in PBS/DMF buffer (V/V = 9/1, pH 7.4) at 528 nm with an excitation wavelength of 293 nm.

Fig. 7
Fig. 7 Images of A549 cells co-cultured with 10 mM PPF-CDNB for 30 minutes.(a) Bright field.(b) Green channel.(c) Overlay.(d-f) Bright field, green channel, and overlay images of A549 cells treated with 50 mM NaHS.(g-i) Bright field, green channel, and overlay images of A549 cells treated with 50 mM Cys. (j-l) Bright field, fluorescence images, and overlay of A549 cells treated with 200 mM PPG. (m-o) Bright field, green channel, and overlay images of A549 cells treated with 200 mM PPG followed by 50 mM Cys.